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Epithelial-mesenchymal transition regulated by EphA2 contributes to vasculogenic mimicry formation of head and neck squamous cell carcinoma.

Wang W, Lin P, Sun B, Zhang S, Cai W, Han C, Li L, Lu H, Zhao X - Biomed Res Int (2014)

Bottom Line: The SiRNA technique was used to knock down the expression of EphA2 in vitro.Knocking down EphA2 in vitro leads to disabled channel-like structure formation, reduction of invasion and migration ability, and reverse of EMT-related markers.Both semiquantitative real-time RT-PCR and immunohistochemistry showed that expressions of EphA2, Twist, and Vimentin were higher in the VM-positive group than in the VM-negative group significantly, while expressions of E-cadherin, claudin4, and DSG-3 were reverse.

View Article: PubMed Central - PubMed

Affiliation: Department of Otorhinolaryngology Head and Neck, Institute of Otorhinolaryngology, Tianjin First Central Hospital, Tianjin 300192, China ; Department of Pathology, Tianjin Cancer Hospital, Tianjin Medical University, Tianjin 300060, China.

ABSTRACT

Purpose: Vasculogenic mimicry (VM) was related to invasion and metastasis of head and neck squamous cell carcinoma (HNSCC) patients. This study was designed to investigate the role of EphA2 in VM formation of HNSCC.

Methods: The SiRNA technique was used to knock down the expression of EphA2 in vitro. The ability of cell migration and invasion were measured by transwell and wound healing assays; three-dimensional culture was used to detect the ability of channel-like structure formation; Western blot was used to detect the expression of epithelial-mesenchymal transition- (EMT-) related molecules in vitro. Further semiquantitative real-time RT-PCR assays and immunohistochemistry were used to demonstrate expression of EphA2 and EMT-related molecules according to VM presence or not in human tissue.

Results: Knocking down EphA2 in vitro leads to disabled channel-like structure formation, reduction of invasion and migration ability, and reverse of EMT-related markers. Both semiquantitative real-time RT-PCR and immunohistochemistry showed that expressions of EphA2, Twist, and Vimentin were higher in the VM-positive group than in the VM-negative group significantly, while expressions of E-cadherin, claudin4, and DSG-3 were reverse.

Conclusions: EphA2 played a key role in VM formation of HNSCC through regulation of EMT.

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Related in: MedlinePlus

Chemotaxis and invasion assays in HNSCC cell lines. (a) SiEphA2/HNSCC cells, scr/HNSCC cells, and rescued cells induced by EGF appeared in a dose-dependent manner, although the chemotaxis of the siEphA2/HNSCC cells was impaired in response to EphA2 reduction. Chemotaxis assays showed that 1 ng/mL EGF was the optimization concentration for transwell. (b) Invasion assays. There was a remarkably reduced invasion in HNSCC cell lines compared with the control cells and rescued cells (P < 0.05).
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fig4: Chemotaxis and invasion assays in HNSCC cell lines. (a) SiEphA2/HNSCC cells, scr/HNSCC cells, and rescued cells induced by EGF appeared in a dose-dependent manner, although the chemotaxis of the siEphA2/HNSCC cells was impaired in response to EphA2 reduction. Chemotaxis assays showed that 1 ng/mL EGF was the optimization concentration for transwell. (b) Invasion assays. There was a remarkably reduced invasion in HNSCC cell lines compared with the control cells and rescued cells (P < 0.05).

Mentions: Another method is chemotaxis referring to directional cell movement dependent on a concentration gradient. Robust chemotaxis of the siEphA2/HNSCC cells, scr/HNSCC cells, and rescued cells induced by EGF appeared in a dose-dependent manner, although the chemotaxis of the siEphA2/HNSCC cells was impaired in response to EphA2 reduction. These results revealed that EphA2 was required for HNSCC cell chemotaxis, while a concentration gradient was required for efficient migration in response to EGF (Figure 4(a)). Subsequently, Boden chamber invasion assays were utilized to confirm the three HNSCC lines' different invasive capabilities after EphA2 knockdown. Knockdown of EphA2 in cell lines significantly reduced the invasion capability compared with the control cells (Figure 4(b)), while overexpression of EphA2 retrieved the ability of invasion in all cell lines. Both results indicated that EphA2 knockdown may decrease migration and invasion capabilities of HNSCC cells. We speculated from our study that VM contributed to invasion and metastasis of HNSCC, and EphA2 may play a key role in this process.


Epithelial-mesenchymal transition regulated by EphA2 contributes to vasculogenic mimicry formation of head and neck squamous cell carcinoma.

Wang W, Lin P, Sun B, Zhang S, Cai W, Han C, Li L, Lu H, Zhao X - Biomed Res Int (2014)

Chemotaxis and invasion assays in HNSCC cell lines. (a) SiEphA2/HNSCC cells, scr/HNSCC cells, and rescued cells induced by EGF appeared in a dose-dependent manner, although the chemotaxis of the siEphA2/HNSCC cells was impaired in response to EphA2 reduction. Chemotaxis assays showed that 1 ng/mL EGF was the optimization concentration for transwell. (b) Invasion assays. There was a remarkably reduced invasion in HNSCC cell lines compared with the control cells and rescued cells (P < 0.05).
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4016880&req=5

fig4: Chemotaxis and invasion assays in HNSCC cell lines. (a) SiEphA2/HNSCC cells, scr/HNSCC cells, and rescued cells induced by EGF appeared in a dose-dependent manner, although the chemotaxis of the siEphA2/HNSCC cells was impaired in response to EphA2 reduction. Chemotaxis assays showed that 1 ng/mL EGF was the optimization concentration for transwell. (b) Invasion assays. There was a remarkably reduced invasion in HNSCC cell lines compared with the control cells and rescued cells (P < 0.05).
Mentions: Another method is chemotaxis referring to directional cell movement dependent on a concentration gradient. Robust chemotaxis of the siEphA2/HNSCC cells, scr/HNSCC cells, and rescued cells induced by EGF appeared in a dose-dependent manner, although the chemotaxis of the siEphA2/HNSCC cells was impaired in response to EphA2 reduction. These results revealed that EphA2 was required for HNSCC cell chemotaxis, while a concentration gradient was required for efficient migration in response to EGF (Figure 4(a)). Subsequently, Boden chamber invasion assays were utilized to confirm the three HNSCC lines' different invasive capabilities after EphA2 knockdown. Knockdown of EphA2 in cell lines significantly reduced the invasion capability compared with the control cells (Figure 4(b)), while overexpression of EphA2 retrieved the ability of invasion in all cell lines. Both results indicated that EphA2 knockdown may decrease migration and invasion capabilities of HNSCC cells. We speculated from our study that VM contributed to invasion and metastasis of HNSCC, and EphA2 may play a key role in this process.

Bottom Line: The SiRNA technique was used to knock down the expression of EphA2 in vitro.Knocking down EphA2 in vitro leads to disabled channel-like structure formation, reduction of invasion and migration ability, and reverse of EMT-related markers.Both semiquantitative real-time RT-PCR and immunohistochemistry showed that expressions of EphA2, Twist, and Vimentin were higher in the VM-positive group than in the VM-negative group significantly, while expressions of E-cadherin, claudin4, and DSG-3 were reverse.

View Article: PubMed Central - PubMed

Affiliation: Department of Otorhinolaryngology Head and Neck, Institute of Otorhinolaryngology, Tianjin First Central Hospital, Tianjin 300192, China ; Department of Pathology, Tianjin Cancer Hospital, Tianjin Medical University, Tianjin 300060, China.

ABSTRACT

Purpose: Vasculogenic mimicry (VM) was related to invasion and metastasis of head and neck squamous cell carcinoma (HNSCC) patients. This study was designed to investigate the role of EphA2 in VM formation of HNSCC.

Methods: The SiRNA technique was used to knock down the expression of EphA2 in vitro. The ability of cell migration and invasion were measured by transwell and wound healing assays; three-dimensional culture was used to detect the ability of channel-like structure formation; Western blot was used to detect the expression of epithelial-mesenchymal transition- (EMT-) related molecules in vitro. Further semiquantitative real-time RT-PCR assays and immunohistochemistry were used to demonstrate expression of EphA2 and EMT-related molecules according to VM presence or not in human tissue.

Results: Knocking down EphA2 in vitro leads to disabled channel-like structure formation, reduction of invasion and migration ability, and reverse of EMT-related markers. Both semiquantitative real-time RT-PCR and immunohistochemistry showed that expressions of EphA2, Twist, and Vimentin were higher in the VM-positive group than in the VM-negative group significantly, while expressions of E-cadherin, claudin4, and DSG-3 were reverse.

Conclusions: EphA2 played a key role in VM formation of HNSCC through regulation of EMT.

Show MeSH
Related in: MedlinePlus